Chymosin is a milk-clotting enzyme that derives from the stomach of mammals where it is produced together with other milk-clotting enzymes, such as pepsin. Preparations of milk-clotting enzymes are used in food industry, for instance cheese manufacture. Thus there is a need for purified chymosin preparations having a high and well characterized specific milk-clotting activity.
Originally chymosin was purified from extracts of bovine stomach, in particular of calves. One main problem with this kind of natural sources have been that the extracts will contain also other milk-clotting enzymes, such as pepsin, and also various proforms of the enzymes. Accordingly earlier purification protocols included steps for transforming proforms to active enzymes and steps separating chymosin from pepsin. Another main problem has been that the activity of the starting material has varied significantly.
Separations of chymosin from pepsin has primarily utilized ion exchange adsorption combined with the fact that there is a large difference in isoelectric points for pepsin and chymosin (pl 2 and pl 4.8, respectively). In other words a cation exchanger which can be negatively charged in the interval pH 2-4.8 will be able to adsorb chymosin in this pH range. In the analogous fashion an anion exchanger which can be positively charged in the same pH interval will be capable of adsorbing chymosin. This has been utilized for instance in U.S. Pat. No. 5,888,966 (Larsen et al) and U.S. Pat. No. 4,745,063 (Birschbach). Affinity adsorption based on dye affinity ligands has also been suggested in U.S. Pat. No. 4,666,843 (Subramanian et al)
Chymosin has also been obtained by so called recombinant techniques, i.e. from host cells that have been transformed to produce chymosin (or proforms thereof). In this case other purification problems arise because the contaminants are not the same, for instance pepsin is lacking (unless the host cell also produce pepsin) and other contaminants have to be removed. Typical host cells can be of microbial origin, such as yeast, fungi (in particular Asperigillus niger), bacteria etc without exclusion of mammalian cells. For this kind of chymosin U.S. Pat. No. 4,743,551 (Subramanian) and U.S. Pat. No. 4,721,673 (Subramanian et al) propose dye affinity ligand adsorption and U.S. Pat. No. 5,122,467 (Heinsohn et al) proposes adsorption to phenyl SEPHAROSE® matrix (SEPHAROSE® is the trade mark of Amersham Pharmacia Biotech. The corresponding products are based on agarose). U.S. Pat. No. 5,122,467 (column 3, lines 31-50) suggests that a comparison has been made between phenyl SEPHAROSE® matrix and agaroses having other functionalities, including octyl. The conclusion stated is that the phenyl functionality is the only one providing the required selectivity for chymosin in fermentation broths. Experiments have also been presented to use other ligands containing aromatic rings in WO 9600735 (Burton et al) and WO 9609116 (Burton et al).
In recent study be Burton et al (“One-step purification of chymosin by mixed mode chromatography” in Biotech. Bioengin. 56(1) (1997) 45-55) a number of chargeable and non-chargeable aromatic ligands have been examined for adsorption of chymosin from a fermentation broth.
In spite of the number of previously suggested purification protocols there is still a need for improvements relating to yield/recovery, purity, specific chymosin activity, simplicity of operation, need for elution agents etc.